This invention relates to adenoviral vectors. More particularly, this invention relates to adenoviral vectors which may be employed in the treatment of hemophilia.
Hemophilias A and B are X-linked, recessive bleeding disorders caused by deficiencies of clotting Factors VIII and IX, respectively. In the United States there are approximately 17,000 patients with hemophilia A and 2,800 with hemophilia B. The clinical presentations for both hemophilias are characterized by episodes of spontaneous and prolonged bleeding. Patients frequently suffer joint bleeds which lead to a disabling arthropathy. Current treatment is directed at stopping the bleeding episodes with intravenous infusions of plasma-derived clotting factors or, for hemophilia A, recombinant Factor VIII. However, therapy is limited by the availability of clotting factors, their short half-lives in vivo, and the high cost of treatment, which can approach 100,000 dollars per year.
Gene therapy offers the promise of a new method of treating hemophilia. Several groups of researchers have conducted research with retroviral vectors containing RNA encoding Factor VIII and Factor IX. Prior to Applicants"" invention, virtually every attempt to produce therapeutic levels of these factors in vivo with such vectors, however, has been unsuccessful. The cDNA and the RNA for Factor VIII has been particularly difficult to work with.
Hoeben et al., J. Biol. Chem, Vol. 265, pgs 7318-7323 (1990) and Israel et al. Blood, Vol. 75, No. 5, pgs. 1074-1080 (Mar. 1, 1990) describe the infection of mouse fibroblasts in vitro with retroviral vectors including DNA (RNA) encoding B-domain deleted human Factor VIII. Although such infected cells were found to express functional human Factor VIII in vitro, the protein was expressed at low levels.
Recently, Hoeben et al., Human Gene Therapy, Vol. 4, pgs 179-186 (1993) infected fibroblasts with retroviral vectors including DNA encoding human Factor VIII. These cells then were implanted into immune-deficient mice. Although cells recovered from the implants up to 2 months post-implantation still had the capacity to secrete Factor VIII when regrown in tissue culture, human Factor VIII was not detected in plasma samples of the recipient mice.
Lynch et al., Human Gene Therapy, Vol. 4, pgs. 259-272 (1993), describes the transfection of PE501 packaging cells with the plasmid forms of retroviral vectors including human Factor VIII cDNA. The virus was harvested, and used to infect PA317 amphotropic retrovirus packaging cells. The infected cells, however, produced human Factor VIII and virus titer in an amount which was about two orders of magnitude lower than those from similar retroviral vectors containing other cDNAs. Lynch et al. also observed a 100-fold lower accumulation of vector RNAs containing the human Factor VIII sequences in comparison to vectors containing other cDNA sequences.
Lynch et al. also reported the following difficulties in working with Factor VIII. High titer human Factor VIII-containing retroviral vector stocks are difficult to generate, and retroviral vectors containing Factor VIII cDNA sequences tend to rearrange and/or delete portions of the Factor VIII cDNA sequences. In addition, Factor VIII mRNA is inherently unstable. Also, the B-domain deleted Factor-VIII coding region contains a 1.2 kb RNA accumulation inhibitory signal.
Thus, there have been significant problems in working with retroviral approaches to gene therapy with Factor VIII and that only limited expression has been achieved prior to Applicants"" invention.
Researchers also have experienced significant difficulties in attempting to achieve therapeutic levels of Factor IX expression with retroviral vectors prior to Applicants"" invention.
Palmer et al., Blood, Vol. 73, No. 2, pgs. 438-445 (February 1989) discloses the transduction of human skin fibroblasts with retroviral vectors including DNA (RNA) encoding human Factor IX. Such transformed fibroblasts then were given to rats and to nude mice. Although such fibroblasts were found to transiently express human Factor IX in the animal blood in amounts up to 190 ng/ml, this amount is not generally considered to be at a therapeutic level.
Scharfmann et al., Proc. Nat. Acad. Sci., Vol. 88, pgs. 4626-4630 (June 1991) discloses the transduction of mouse fibroblast implants with a retroviral vector including a B-galactosidase gene under the control of the dihydrofolate reductase (DHFR) promoter. Such fibroblasts then were grafted into mice, and expression of the xcex2-galactosidase gene for up to sixty days was obtained. Scharfmann et al. also disclose fibroblasts transduced with canine Factor IX, but they only obtained short-term and non-therapeutic levels of expression.
Dai et al., Proc. Nat. Acad. Sci., Vol. 89, pgs. 10892-10895 (November 1992) discloses the transfection of mouse primary myoblasts with retroviral vectors including canine Factor IX DNA under the control of a mouse muscle creatine kinase enhancer and a human cytomegalovirus promoter. The transfected myoblasts then were injected into the hind legs of mice. Expression of canine Factor IX over a period of 6 months was obtained; however, the steady-state levels of Factor IX secreted into the plasma (10 ng/ml for 107 injected cells) are not sufficient to be of therapeutic value.
Gerrard et al., Nature Genetics, Vol. 3, pgs. 180-183 (February 1993), discloses the transfection of primary human keratinocytes with a retroviral vector including a human Factor IX gene under the control of the retroviral LTR. The transformed keratinocytes then were transplanted into nude mice, and human Factor IX was detected in the bloodstream for about 1 week. The amounts of Factor IX, however, were about 2.5 ng/ml, or about 1% of a therapeutic dose.
Kay et al., Science, Vol. 262, pgs. 117-119 (Oct. 1, 1993) discloses the direct infusion of retroviral vectors including Factor IX DNA into the portal vasculature of dogs following partial hepatectomy. The animals expressed low levels of canine Factor IX for more than 5 months. Although such expression of Factor IX resulted in reductions of whole blood clotting and partial thromboplastin times of the treated animals, the authors stated that increased levels of Factor IX must first be achieved before the technique could be applied to humans.
Zhou et al., Science in China, Vol. 36, No. 9, pgs. 33-41 (September 1993) discloses the transfection of rabbit skin fibroblasts with retroviral vectors including DNA encoding human Factor IX. The fibroblasts then were implanted into rabbits as autografts or allografts. Expression of the human Factor IX was maintained in the rabbits for over 10 months. Factor IX levels in the rabbit plasma of up to 480 ng/ml were claimed to have been achieved; however, the assay used to measure Factor IX employed an anti-rabbit antibody that had the potential of generating false positive results.
Lu et al., Science in China, Vol. 36, No. 11, pgs. 1341-1351 (November 1993) and Hsueh et al., Human Gene Therapy, Vol. 3, pgs. 543-552 (1992) discloses a human gene therapy trial in which human skin fibroblasts were taken from two hemophiliac patients, and transfected with retroviral vectors including DNA encoding human Factor IX. The cells then were pooled and embedded in a collagen mixture, and the cells then were injected into the patients. In one patient, the concentration of human Factor IX increased from 71 ng/ml to 220 ng/ml, with a maximum level of 245 ng/ml. The clotting activity of this patient increased from 2.9% to 6.3% of normal. In the other patient, the plasma level of Factor IX increased from 130 ng/ml to 250 ng/ml, and was maintained at a level of 220 ng/ml for 5xc2xd months; however, the clotting activity has not increased. Lack of pretreatment Factor IX data on these patients makes it difficult to interpret the small increases in Factor IX seen in treatment.
The conclusion to be drawn from scientific literature at the time of Applicants"" invention, on the attempts to use retroviruses in gene therapy for hemophilia A and hemophilia B is that, in spite of a very concerted effort and numerous attempts, by and large the field has failed to produce retroviral vectors that can be used to achieve therapeutic levels of expression of human Factor VIII or human Factor IX in vivo. Working with Factor VIII has been especially difficult, and the results have been unsatisfactory. The experimental strategies described above are laborious and clinically invasive.
Adenoviral vectors offer another approach to gene therapy. Adenovirus genomes are linear, double-stranded DNA molecules of approximately 36 kilobase pairs. Each extremity of the viral genome has a short sequence known as the inverted terminal repeat (or ITR), which is necessary for viral replication. The well-characterized molecular genetics of adenovirus render it an advantageous vector for gene transfer. Portions of the viral genome can be substituted with DNA of foreign origin. In addition, recombinant adenoviruses are structurally stable and no rearranged viruses are observed after extensive amplification.
Recombinant adenoviruses have been used as efficient vectors for gene transfer into a number of cell types. There are several reports of hepatocyte transduction: Jaffe et al., Nature Genetics, Vol. 1, pgs. 372-378 (1992) (alpha-1-antitrypsin); Li et al., Human Gene Therapy, Vol. 4, pgs. 403-409 (1993) (beta-galactosidase); Stratford-Perricaudet et al., Human Gene Therapy, Vol. 1, pgs. 241-256 (1990) (ornithine transcarbamylase); Smith, et al., Nature Genetics, Vol. 5, pgs. 397-402 (1993) (Factor IX); and J. Am. Med. Assoc., Vol. 269, No. 7, pg. 838 (Feb. 17, 1993) (marker protein).
Because Factor VIII is synthesized largely in hepatocytes (Kelly et al. Br. J. Haemat., Vol. 56, pgs. 535-543 (1984); Wion et al., Nature, Vol. 317, pgs. 726-729 (1985); Zelechovska et al. Nature, Vol. 317, pgs. 729-732 (1985)), transduction of hepatocytes with a Factor VIIIxe2x80x94containing recombinant adenovirus, resulting in the expression of Factor VIII protein in vivo, may be an effective gene therapy-based treatment for hemophilia A.
The inventors have discovered how to produce high titer, stable, adenoviral vectors that produce therapeutic levels of clotting factors when administered to an animal host. These vectors mediate gene transfer in vivo and will enable treatment protocols to be much less laborious and invasive than those previously described.